Applications in Forensic Proteomics: Protein Identification and Profiling......Page 2
Applications in Forensic Proteomics: Protein Identification and Profiling......Page 4
Library of Congress Cataloging-in-Publication Data......Page 5
Foreword......Page 6
Unambiguous Identification of Ricin and Abrin with Advanced Mass Spectrometric Assays......Page 8
Subject Index......Page 9
The Success of Proteomics in the Biological Sciences......Page 10
Why Analyze Proteins?......Page 11
Human Forensic Proteomics......Page 12
Protein Toxins......Page 13
The Future......Page 14
References......Page 15
What is Proteomics?......Page 18
Liquid Chromatography......Page 20
Mass Spectrometry......Page 21
Tandem Mass Spectrometry and Peptide Fragmentation......Page 22
Targeted versus Untargeted Methods......Page 23
Figure 2. Annotated theoretical (A) and observed (B) mass spectrum of the peptide LEQLAGNLR, derived from the protein ricin, acquired during an LC-MS/MS analysis using a Thermo Scientific Q Exactive HF mass spectrometer 10. Peak spacing corresponding to amino acid masses are shown by dotted arrows. Unassigned peaks are in black, y-ions are in blue, b-ions are in red, and immonium ions are in magenta. Satellite peaks due to neutral loss of water or ammonia are also shown in (B).......Page 24
Peptide Identification by Tandem Mass Spectrometry......Page 25
Spectral Library Search......Page 26
De Novo Peptide Identification......Page 27
Sequence Tag-Based Peptide Identification......Page 28
Quality Control of Peptide Identification Results: PSM Filtering and the False Discovery Rate......Page 29
Protein Inference......Page 30
Protein Quantitation......Page 31
Legal Aspects—Admissibility of Evidence......Page 32
References......Page 33
Proteomic Sample Preparation Techniques: Toward Forensic Proteomic Applications......Page 38
Figure 1. Proteomic sample dependent pretreatment analysis workflow. Possible sample pretreatment steps A-D and subsequent processing steps prior to mass spectrometry analyses. Offline or online fractionation can also be utilized prior to LC-MS/MS analysis for in-depth proteome coverage.......Page 39
Potential Pretreatment Steps......Page 40
Powder or No-Lysis Sample (Figure 1D)......Page 41
In-Solution Protein Digestion......Page 42
High Performance Liquid Chromatography (HPLC)......Page 43
Method Details – Options For Each Step......Page 44
Acknowledgments......Page 49
References......Page 50
NextGen Serology: Leveraging Mass Spectrometry for Protein-Based Human Body Fluid Identification......Page 56
The Investigative Value of Forensic Serology in the Era of DNA Profiling......Page 57
Enzyme Activity Assays......Page 58
Figure 1. Examples of results obtained from presumptive testing for the presence of blood using the Kastle–Meyer (phenolphthalein) assay. The swab on the top illustrates a negative result while the swab on the bottom shows the characteristic deep pink color, which exemplifies a strong positive assay result.......Page 59
Light/Fluorescence Microscopy......Page 60
Limitations of Existing Serological Assays......Page 61
Messenger RNA and MicroRNA as Biomarkers......Page 63
Spectroscopic Signatures as Biomarkers......Page 65
Applications of Advanced Proteomic Technologies to Forensic Science......Page 66
Biomarker Discovery Comparative Proteomics......Page 67
Protein Biomarker Verification for Serological Testing......Page 69
Figure 3. Example of the results obtained from the analysis of a vaginal fluid swab using a targeted-ion assay that scans for biomarker peptides for vaginal secretions, menstrual fluid, seminal fluid, urine, peripheral blood and saliva. (Top) Q-TOF multiplex data indicating the detection of vaginal fluid biomarker peptides. (Bottom) Database search results indicating a high-confidence confirmatory identification of human vaginal fluid. Only the targeted vaginal fluid biomarkers were detected. No biomarkers indicative of any other body fluids were detected.......Page 70
Proteomic Assay Validation......Page 71
Figure 4. Overview of the workflow for Q-TOF body fluid identification and STR profiling from a mixture of seminal fluid and saliva. (1) The dried stain was located by use of an alternative light source. (2) A cutting was rehydrated in phosphate buffered saline and placed in a filter basket to recover the retained fluid from which DNA was isolated. (3) The recovered DNA was quantified using the Applied Biosystems® Quantifiler® Duo kit (Thermo Fisher Scientific™) and total protein concentration was determined using the Micro BCA™ Protein Assay Kit (Thermo Fisher Scientific™). (4) The DNA-depleted extract wash was processed for confirmatory body fluid identification using the Q-TOF multiplex assay. A full DNA profile was generated from the DNA extract using the Applied Biosystems® AmpFlSTR® Identifiler® Plus PCR Amplification Kit (Thermo Fisher Scientific™).......Page 74
Study Findings......Page 75
Sexual Assault Kit Screening and Sample Prioritization......Page 78
Study Findings......Page 79
Future Directions......Page 81
Figure 5. Detection of the semenogelin-2 peptide GSISIQTEEK extracted from vaginal swabs spiked with three different quantities of semen. The resulting response for iMRM purified swabs (top) allowed for the unambiguous identification of a high-specificity seminal fluid biomarker. The same semenogelin-2 peptide was not readily detected in replicate swabs prepared using the standard (i.e., non-enriched) sample preparation protocol (bottom).......Page 82
References......Page 83
The Importance of Body Fluid Identification in the Era of Forensic DNA Analysis......Page 90
Body Fluid Marker Proteins & Body Fluid Mixtures......Page 91
Menstrual Blood Marker Proteins......Page 93
Informatic Approaches for Menstrual Blood Identification Using the Relative Abundance of Many Proteins......Page 94
Figure 1. We used liquid chromatography mass spectrometry and isobaric labeling to measure the proteome of 45 venous and 126 menstrual blood samples. After identifying and quantifying the peptides, their quantities were normalized in two steps: (i) using a standard sample in each of the batches and (ii) median centering each sample to correct for differences in the total amount of protein loaded. Example of two peptide that are commonly observed in both venous and menstrual blood samples, and their intensities separates the venous and menstrual blood samples well but not perfectly (Left and Middle Panels). The two peptide intensities combined gives a better separation, although still not a perfect separation (Right Panel).......Page 95
Deconvoluting Mixtures of Body Fluids......Page 96
References......Page 97
Introduction......Page 100
Fingermark Proteomics......Page 102
Figure 1. MALDI MS Profiling of an unwashed male natural mark showing (intact) peptides and proteins.......Page 103
Figure 2. MALDI MS Profiling and Imaging of peptides/small proteins in a latent fingermark. Half (Panel A) of a split mark was prepared using the dried-droplet method spotting 5 mg/mL CHCA in 25:25:50 acetonitrile/ethanol/0.5% TFA. Other fingermark half (Panel B) was prepared using the same matrix solution but sprayed using the SunCollect autospraying system (Sunchrom, Germany) showing a rather decreased peptide/protein ion signal intensity. Panel (C) shows the image of a latent mark prepared using the dry-wet method 32 by dusting with CHCA and spraying a 70:30 ACN/ 0.5% TFA solution using a SunCollect autosprayer and acquired on a more sensitive instrument. The speckled image of a peptide at m/z 3369 is shown. Adapted and reproduced with permission from reference 22 Copyright (2016) Springer Nature.......Page 105
Figure 3. MALDI MS spectrum of in situ proteolysis of a fingermark. Trypsin was spotted on top the fingermark which was then incubated at 50°C for 2 hr in a K2SO4 saturated atmosphere. The spectrum shows a large abundance of putatively assigned keratin ion signals with high mass accuracy (only the most abundant have been labeled).......Page 107
In Situ Proteomics and Peptide Imaging in Blood Marks......Page 108
Figure 5. MALDI MS Imaging analysis of a blood fingermark. The mark was split in quarters and spray coat using different trypsin concentrations prior to matrix application. a. blood mark; b. blood mark quarters after in situ proteolysis and matrix spray coat (1 quarter is missing as at the highest trypsin concentration, the high viscosity determined a capillary blockage); c-d. blood protein mapping through peptide derived peptides from Complement C3, α- Hemoglobin, hemopexin and serotransferrin. Adapted and reproduced with permission from 37 Copyright (2016) John Wiley and Sons.......Page 109
Conclusions......Page 111
References......Page 112
Human Identification Using Genetically Variant Peptides in Biological Forensic Evidence......Page 116
Protein-Based Human Identification......Page 117
Figure 1. Illustration of an nsSNP in a DNA sequence and the corresponding SAP in the protein that is coded by that DNA sequence. The sequence on the left shows the reference sequence and three codons that encode for three amino acids. A SNP in the variant sequence on the right encodes for a different amino acid in the protein.......Page 118
Exome-Driven Approach to GVP Discovery......Page 119
Mass Spectrometry Considerations in GVP Discovery......Page 120
Figure 3. Comparison of identified proteins between single one-inch and bulk (10 mg) quantities of scalp hair. Data are from reference 22.......Page 121
Figure 4. Distribution of identified proteins in modern and archaeological hair shaft specimens by protein function. Reproduced with permission from reference 17. Creative Commons CC0.......Page 122
Bone and Skin Proteome......Page 123
Figure 5. Subset of differentially expressed proteins among scalp, arm, and pubic hairs. Adapted from reference 24 with permission. Creative Commons BY.......Page 124
Figure 7. Distribution of proteins in rib bone tissue by biological function. Data are from reference 18.......Page 125
Translating GVP Technology for Forensic Investigations......Page 126
Figure 8. Random match probabilities of single and bulk hair samples as a function of the number of inferred SNPs from GVPs. Two single-hair sample replicates are labeled. Data are from reference 22.......Page 127
Conclusion......Page 128
Figure 9. Workflow of a general GVP identification approach for comparison of forensic hair evidence to a suspect’s DNA. GVPs from forensic evidence are identified via targeted liquid chromatography-tandem mass spectrometry given a list of targets from the general GVP panel. Predicted GVPs from a suspect’s DNA are compared to GVPs identified from hair evidence. In this scenario, both the suspect and the individual to which the evidence belong are heterozygotes for the SNP rs1732263 in K82, as both the non-mutated and SAP-containing forms of the GVP are identified.......Page 129
References......Page 130
Introduction......Page 134
Figure 1. Example spectra of peptide mass fingerprints from an ancient bear (top) and ancient human (bottom) bone sample showing selected peptides annotated with their respective sequence differences (smal lettering used to highlight amino acid substitutions).......Page 136
Bone Structure......Page 137
Methodologies for the Analysis of Bone Proteomes......Page 139
Ancient and Forensic Proteins for Species Identification......Page 140
Figure 3. Typical protein identification via peptide spectra matching showing example of pig bone (from 34) showing (A) good sequence coverage (>50%), (B) an example of the interpretation of a peptide spectrum match informing the score given, and (C) comparison of this particular peptide sequence with those of other animals.......Page 141
Figure 4. Normalized protein abundances for the three proteins of interest that increase (A1AT & CMGA) or decrease (FETUA) with biological age, with their 3D structures.......Page 142
Proteome Changes with Length of Burial Time......Page 143
Future Directions of Forensic Bone Proteomics and Concluding Remarks......Page 144
References......Page 145
Introduction......Page 152
Microbial Identification by Targeted Proteomics......Page 153
Microbial Identification with MALDI-TOF MS......Page 154
Database Selection......Page 156
Choice of Peptides to Use in Identification Algorithms......Page 157
Decision Criteria......Page 158
Future Directions: Increasingly Complex Mixtures......Page 159
Distinguishing Laboratory-Adapted or Laboratory-Grown Pathogenic Bacteria from Wild Isolates: Serial Passaging of Yersinia pestis Wild Isolates and Classification by Machine Learning......Page 160
Characterizing Microbiological Growth Medium Components......Page 162
Determining the Host Cell Species of a Virus Preparation......Page 163
References......Page 164
ISO 17025 Accreditation of Method-Based Mass Spectrometry for Bioforensic Analyses......Page 170
Ricinus communis Toxin......Page 171
Ricin Characterization Methods Workflow......Page 172
MS Methods Maintenance......Page 173
Sample Preparation......Page 174
MW Estimation by SDS-PAGE......Page 175
Figure 3. Four Parameter Logistic Regression Curves for Volume Measurements. Mean volume (relative units based on gel band pixel density) measurements plotted against ricin concentrations from 6 SDS-PAGE replicates. Volume measurements obtained from NRl bands for ricin, imaged using Gel Doc™ EZ and Image Lab™ software (Bio-Rad). Data fit to a 4 parameter logistic curve using displayed equations. Error bars represent ± SD. Limit of detection (LOD) estimated for ricin (1.2 µg/mL) represented by dotted line.......Page 176
Results......Page 177
Results......Page 179
References......Page 182
Introduction......Page 184
Targeted MS/MS......Page 186
Proteomic MS/MS......Page 187
Measuring Ricin’s Activity with Mass Spectrometry......Page 188
Figure 2. Detection of the cleavage product from RNA14 by various concentrations of ricin after incubation for 0.5 h (A) and 4 h (B). The toxin was spiked in 0.5 mL of PBST buffer (●) or 2% milk (○) followed by enrichment with specific antibodies immobilized on magnetic beads. (C). Some typical mass spectra obtained from the aliquots of the ricin activity reaction (55 °C, 4 h). Reproduced with permission from reference 24. Copyright 2016 American Chemical Society.......Page 189
Conclusions......Page 190
References......Page 191
Challenges in the Development of Reference Materials for Protein Toxins......Page 194
Biological Toxins......Page 195
On the Importance of Quality-Control Tools......Page 196
Figure 1. General procedure for CRM production applied at JRC Geel 53.......Page 199
Challenges in the Development of CRMs for Protein Toxins......Page 200
Characterization of Toxin CRMs......Page 203
Special Consideration of the Use of Toxin CRMs in Mass Spectrometry......Page 204
Abbreviations......Page 205
References......Page 206
The Challenge of Modern Forensics......Page 212
Reliability and Relevance: The Foundation for a Defensible Method......Page 214
Questions an Investigator Might Ask......Page 215
Figure 1. The forensic method development process.......Page 216
Seven Elements of a Defensible Method......Page 217
A Standard Data Analysis and Interpretation Protocol......Page 218
Verified and Validated Statistical Performance Criteria......Page 220
An Interpretation Framework for Evaluating the Strength of Evidence......Page 221
Clearly Stated Operational Limitations......Page 222
Peer-Reviewed Publication......Page 223
Additional Guidelines for Forensic Proteomics......Page 224
Guidelines Relevant to All Forensic Proteomics Assays......Page 225
Additional Guidelines for Untargeted Assays......Page 226
References......Page 228
Eric D. Merkley......Page 238
Indexes......Page 240
Author Index......Page 242
F......Page 244
G......Page 245
P......Page 246